Reovirus for the treatment of neoplasia

ABSTRACT

Methods for treating neoplasia, by administering reovirus to a Ras-mediated neoplasm, and use of reovirus for manufacture of a medicament for the treatment of neoplasia, are disclosed. The reovirus is administered so that it ultimately directly contacts cells of the neoplasm. Human reovirus, non-human mammalian reovirus, and/or avian reovirus can be used. If the reovirus is human reovirus, type 1 (e.g., strain Lang), type 2 (e.g., strain Jones), type 3 (e.g., strain Dearing or strain Abney), as well as other sterotypes or strains of reovirus can be used. Combinations of more than one type and/or strain or reovirus can be used, as can reovirus from different species of animal. Either solid neoplasms or hematopoietic neoplasms can be treated.

RELATED APPLICATION

[0001] This application is a Continuation-in-Part of U.S. Ser. No.08/911,383 filed on Aug. 13, 1997, the entire teachings of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Normal cell proliferation is regulated by a balance betweengrowth-promoting proto-oncogenes and growth-constrainingtumor-suppressor genes. Tumorigenesis can be caused by geneticalterations to the genome that result in the mutation of those cellularelements that govern the interpretation of cellular signals, such aspotentiation of proto-oncogene activity or inactivation of tumorsuppression. It is believed that the interpretation of these signalsultimately influences the growth and differentiation of a cell, and thatmisinterpretation of these signals can result in neoplastic growth(neoplasia).

[0003] Genetic alteration of the proto-oncogene Ras is believed tocontribute to approximately 30% of all human tumors (Wiessmuller, L. andWittinghofer, F. (1994), Cellular Signaling 6(3):247-267; Barbacid, M.(1987) A Rev. Biochem. 56, 779-827). The role that Ras plays in thepathogenesis of human tumors is specific to the type of tumor.Activating mutations in Ras itself are found in most types of humanmalignancies, and are highly represented in pancreatic cancer (80%),sporadic colorectal carcinomas (40-50%), human lung adenocarcinomas(15-24%), thyroid tumors (50%) and myeloid leukemia (30%) (Millis, Nebr.et al. (1995) Cancer Res. 55:1444; Chaubert, P. et al. (1994), Am. J.Path. 144:767; Bos, J. (1989) Cancer Res. 49:4682). Ras activation isalso demonstrated by upstream mitogenic signaling elements, notably bytyrosine receptor kinases (RTKs). These upstream elements, if amplifiedor overexpressed, ultimately result in elevated Ras activity by thesignal transduction activity of Ras. Examples of this includeoverexpression of PDGFR in certain forms of glioblastomas, as well as inc-erbB-2/neu in breast cancer (Levitzki, A. (1994) Eur. J. Biochem.226:1; James, P. W., et al. (1994) Oncogene 9:3601; Bos, J. (1989)Cancer Res. 49:4682).

[0004] Current methods of treatment for neoplasia include surgery,chemotherapy and radiation. Surgery is typically used as the primarytreatment for early stages of cancer; however, many tumors cannot becompletely removed by surgical means. In addition, metastatic growth ofneoplasms may prevent complete cure of cancer by surgery. Chemotherapyinvolves administration of compounds having antitumor activity, such asalkylating agents, antimetabolites, and antitumor antibiotics. Theefficacy of chemotherapy is often limited by severe side effects,including nausea and vomiting, bone marrow depression, renal damage, andcentral nervous system depression. Radiation therapy relies on thegreater ability of normal cells, in contrast with neoplastic cells, torepair themselves after treatment with radiation. Radiotherapy cannot beused to treat many neoplasms, however, because of the sensitivity oftissue surrounding the tumor. In addition, certain tumors havedemonstrated resistance to radiotherapy and such may be dependent ononcogene or anti-oncogene status of the cell (Lee. J. M. et al. (1993)PNAS 90:5742-5746; Lowe. S. W. et al. (1994) Science, 266:807-810;Raybaud-Diogene. H. et al. (1997) J. Clin. Oncology, 15(3):1030-1038).In view of the drawbacks associated with the current means for treatingneoplastic growth, the need still exists for improved methods for thetreatment of most types of cancers.

SUMMARY OF TE INVENTION

[0005] The present invention pertains to methods for treating neoplasiain a mammal, using reovirus, and to use of reovirus for manufacture of amedicament for the treatment of neoplasia. Reovirus is administered to aneoplasm, in which an element of the Ras signaling pathway (eitherupstream or downstream) is activated to an extent that results inreovirus-mediated oncolysis of cells of the neoplasm. The reovirus canbe administered in a single dose or in multiple doses; furthermore, morethan one neoplasm in an individual mammal can be treated concurrently.Both solid neoplasms and hematopoietic neoplasms can be targeted. Thereovirus is administered so that it contacts cells of the mammal (e.g.,by injection directly into a solid neoplasm, or intravenously into themammal for a hematopoietic neoplasm). The methods can be used to treatneoplasia in a variety of mammals, including mice, dogs, cats, sheep,goats, cows, horses, pigs, and non-human primates. Preferably, themethods are used to treat neoplasia in humans.

[0006] The methods and uses of the invention provide an effective meansto treat neoplasia, without the side effects associated with other formsof cancer therapy. Furthermore, because reovirus is not known to beassociated with disease, any safety concerns associated with deliberateadministration of a virus are minimized.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1 is a depiction of the molecular basis of reovirusoncolysis, in which the reovirus usurps the host cell Ras signallingpathway.

[0008]FIG. 2 is a graphic representation of the effects over time ofactive (open circles) or inactivated (closed circles) reovirus serotype3 (strain Dearing) on the size of murine THC-11 tumors grown in severecombined immunodeficiency (SCID) mice. The plotted values represent themean of the measurements with the standard error of the mean also shown.

[0009]FIG. 3 is a graphic representation of the effects over time ofactive (open circles) or inactivated (closed circles) reovirus serotype3 (strain Dearing) on the size of human glioblastoma U-87 xenograftsgrown in SCID mice. The plotted values represent the mean of themeasurements with the standard error of the mean also shown.

[0010]FIG. 4 is a graphic representation of the effects over time ofactive (open circles, open squares) or inactivated (closed circles,closed squares) reovirus serotype 3 (strain Dearing) on the size oftreated (circles) or untreated (squares) bilateral human glioblastomaU-87 xenografts grown in SCID mice. The plotted values represent themean of the measurements with the standard error of the mean also shown.

[0011] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The invention pertains to methods of treating a neoplasm in amammal, by administering reovirus to the neoplasm. The name reovirus(Respiratory and enteric orphan virus) is a descriptive acronymsuggesting that these viruses, although not associated with any knowndisease state in humans, can be isolated from both the respiratory andenteric tracts (Sabin, A. B. (1959), Science 130:966). The mammalianreovirus consists of three serotypes: type 1 (strain Lang or TIL), type2 (strain Jones, T2J) and type 3 (strain Dearing or strain Abney, T3D).The three serotypes are easily identifiable on the basis ofneutralization and hemagglutinin-inhibition assays (Sabin, A. B. (1959),Science 130:966; Fields, B. N. et al. (1996), Fundamental Virology, 3rdEdition, Lippincott-Raven; Rosen, L. (1960) Am. J. Hyg. 71:242; Stanley,N. F. (1967) Br. Med. Bull 23:150).

[0013] Although reovirus is not known to be associated with anyparticular disease (Tyler, K. L. and Fields, B. N., in Fields Virology(Fields, B. N., Knipe, D. M., and Howley, P. M. eds), Lippincott-Raven,Philadelphia, 1996, p. 1597), many people have been exposed to reovirusby the time they reach adulthood (i.e., fewer than 25% in children<5years old, to greater than 50% in those 20-30 years old (Jackson G. G.and Muldoon R. L. (1973) J. Infect. Dis. 128:811; Stanley N. F. (1974)In: Comparative Diagnosis of Viral Diseases, edited by E. Kurstak and KKurstak, 385-421, Academic Press, New York).

[0014] For mammalian reoviruses, the cell surface recognition signal issialic acid (Amistrong, G. D. et al. (1984), virology 138:37; Gentsch,J. R. K and Pacitti, A. F. (1985), J. Virol. 56:356; Paul R. W. et al.(1989) Virology 172:382-385). Due to the ubiquitous nature of sialicacid, reovirus binds efficiently to a multitude of cell lines and assuch can potentially target many different tissues; however, there aresignificant differences in susceptibility to reovirus infection betweencell lines.

[0015] As described herein, Applicants have discovered that cells whichare resistant to reovirus infection became susceptible to reovirusinfection when transformed with a gene in the Ras pathway. “Resistance”of cells to reovirus infection indicates that infection of the cellswith the virus did not result in significant viral production or yield.Cells that are “susceptible” are those that demonstrate induction ofcytopathic effects, viral protein synthesis, and/or virus production.Resistance to reovirus infection was found to be at the level of genetranslation, rather than at early transcription: while viral transcriptswere produced, virus proteins were not expressed. Viral genetranscription in resistant cells correlated with phosphorylation of anapproximately 65 kDa cell protein, determined to be double-strandedRNA-activated protein kinase (PKR), that was not observed in transformedcells. Phosphorylation of PKR lead to inhibition of translation. Whenphosphorylation was suppressed by 2-aminopurine, a known inhibitor ofPKR, drastic enhancement of reovirus protein synthesis occurred in theuntransformed cells. Furthermore, in a severe combined immunodeficiency(SCID) mouse model in which tumors were created on both the right andleft hind flanks revealed that reovirus significantly reduced tumor sizewhen injected directly into the right-side tumor, in addition,significant reduction in tumor size was also noted on the left-sidetumor which was not directly injected with reovirus, indicating that theoncolytic capacity of the reovirus was systemic as well as local.

[0016] These results indicated that reovirus uses the host cell's Raspathway machinery to downregulate PKR and thus reproduce. FIG. 1 depictsthe usurpation of the host cell Ras signalling pathway by reovirus. Asshown in FIG. 1, for both untransformed (reovirus-resistant) and EGFR-,Sos-, or ras-transformed (reovirus-susceptible) cells, virus binding,internalization, uncoating, and early transcription of viral genes allproceed normally. In the case of untransformed cells, secondarystructures on the early viral transcripts inevitably trigger thephosphorylation of PKR, thereby activating it, leading to thephosphorylation of the translation initiation factor eIF-2α, and hencethe inhibition of viral gene translation. In the case of EGFR-, Sos-, orras-transformed cells, the PKR phosphorylation step is prevented orreversed by Ras or one of its downstream elements, thereby allowingviral gene translation to ensue. The action of Ras (or a downstreamelement) can be mimicked by the use of 2-aminopurine (2-AP), whichpromotes viral gene translation (and hence reovirus infection) inuntransformed cells by blocking PKR phosphorylation.

[0017] Based upon these discoveries, Applicants have developed methodsfor treating neoplasms in mammals. Representative mammals include mice,dogs, cats, sheep, goats, cows, horses, pigs, non-human primates, andhumans. In a preferred embodiment, the mammal is a human.

[0018] In the methods of the invention, reovirus is administered to aneoplasm in the individual mammal. Representative types of humanreovirus that can be used include type 1 (e.g., strain Lang or T1L);type 2 (e.g., strain Jones or T2J); and type 3 (e.g., strain Dearing orstrain Abney, T3D or T3A); other strains of reoyirus can also be used.In a preferred embodiment, the reovirus is strain Dearing.Alternatively, the reovirus can be a non-human mammalian reovirus (e.g.,non-human primate reovirus, such as baboon reovirus; equine; or caninereovirus), or a non-mammalian reovirus (e.g., avian reovirus). Acombination of different serotypes and/or different strains of reovirus,such as reovirus from different species of animal, can be used. Thereovirus is “naturally-occurring”: that is, it can be isolated from asource in nature and has not been intentionally modified by humans inthe laboratory. For example, the reovirus can be from a “field source”:that is, from a human patient. If desired, the reovirus can bechemically or biochemically pretreated (e.g., by treatment with aprotease, such as chymotrypsin or trypsin) prior to administration tothe neoplasm. Such pretreatment removes the outer coat of the virus andmay thereby result in better infectivity of the virus.

[0019] The neoplasm can be a solid neoplasm (e.g., sarcoma orcarcinoma), or a cancerous growth affecting the hematopoietic system (a“hematopoietic neoplasm”; e.g., lymphoma or leukemia). A neoplasm is anabnormal tissue growth, generally forming a distinct mass, that grows bycellular proliferation more rapidly than normal tissue growth. Neoplasmsshow partial or total lack of structural organization and functionalcoordination with normal tissue. As used herein, a “neoplasm”, alsoreferred to as a “tumor”, is intended to encompass hematopoieticneoplasms as well as solid neoplasms. At least some of the cells of theneoplasm have a mutation in which the Ras gene (or an element of the Rassignaling pathway) is activated, either directly (e.g., by an activatingmutation in Ras) or indirectly (e.g., by activation of an upstreamelement in the Ras pathway). Activation of an upstream element in theRas pathway includes, for example, transformation with epidermal growthfactor receptor (EGFR) or Sos. A neoplasm that results, at least inpart, by the activation of Ras, an upstream element of Ras, or anelement in the Ras signalling pathway is referred to herein as a“Ras-mediated neoplasm”. One neoplasm that is particularly susceptibleto treatment by the methods of the invention is pancreatic cancer,because of the prevalence of Ras-mediated neoplasms associated withpancreatic cancer. Other neoplasms that are particularly susceptible totreatment by the methods of the invention include breast cancer, braincancer (e.g., glioblastoma), lung cancer, prostate cancer, colorectalcancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, andleukemia.

[0020] The reovirus is typically administered in a physiologicallyacceptable carrier or vehicle, such as phosphate-buffered saline, to theneoplasm. “Administration to a neoplasm” indicates that the reovirus isadministered in a manner so that it contacts the cells of the neoplasm(also referred to herein as “neoplastic cells”). The route by which thereovirus is administered, as well as the formulation, carrier orvehicle, will depend on the location as well as the type of theneoplasm. A wide variety of administration routes can be employed. Forexample, for a solid neoplasm that is accessible, the reovirus can beadministered by injection directly to the neoplasm. For a hematopoieticneoplasm, for example, the reovirus can be administered intravenously orintravascularly. For neoplasms that are not easily accessible within thebody, such as metastases or brain tumors, the reovirus is administeredin a manner such that it can be transported systemically through thebody of the mammal and thereby reach the neoplasm (e.g., intrathecally,intravenously or intramuscularly). Alternatively, the reovirus can beadministered directly to a single solid neoplasm, where it then iscarried systemically through the body to metastases. The reovirus canalso be administered subcutaneously, intraperitoneally, topically (e.g.,for melanoma), orally (e.g., for oral or esophageal neoplasm), rectally(e.g., for colorectal neoplasm), vaginally (e.g., for cervical orvaginal neoplasm), nasally or by inhalation spray (e.g., for lungneoplasm).

[0021] The reovirus is administered in an amount that is sufficient totreat the neoplasm (e.g., an “effective amount”). A neoplasm is“treated” when administration of reovirus to cells of the neoplasmeffects oncolysis of the neoplastic cells, resulting in a reduction insize of the neoplasm, or in a complete elimination of the neoplasm. Thereduction in size of the neoplasm, or elimination of the neoplasm, isgenerally caused by lysis of neoplastic cells (“oncolysis”) by thereovirus. The effective amount will be determined on an individual basisand may be based, at least in part, on consideration of the type ofreovirus; the individual's size, age, gender, and the size and othercharacteristics of the neoplasm. For example, for treatment of a human,approximately 10³ to 10¹² plaque forming units (PFU) of reovirus can beused, depending on the type, size and number of tumors present. Thereovirus can be administered in a single dose, or multiple doses (i.e.,more than one dose). The multiple doses can be administeredconcurrently, or consecutively (e.g., over a period of days or weeks).The reovirus can also be administered to more than one neoplasm in thesame individual.

[0022] The invention is further illustrated by the followingExemplification.

EXEMPLIFICATION

[0023] Materials and Methods

[0024] Cells and Virus

[0025] Parental NIH-3T3 cell lines along with NIH-3T3 cells transformedwith a number of oncogenes were obtained from a variety of sources.Parental NIH-3T3 and NIH-3T3 cells transfected with the Harvey-ras(H-ras) and EJ-ras oncogenes were a generous gift of Dr. Douglas Faller(Boston University School of Medicine). NIH-3T3 cells along with theirSos-transformed counterparts (designated TNIH#5) were a generous gift ofDr. Michael Karin (University of California, San Diego). Dr. H.-J. Kung(Case Western Reserve University) kindly donated parental NIH-3T3 cellsalong with NIH-3T3 cells transfected with the v-erbB oncogene(designated THC-11). 2H1 cells, a derivative of the C3H 10T1/2 murinefibroblast line, containing the Harvey-ras gene under thetranscriptional control of the mouse metallothionein-I promoter wereobtained from Dr. Nobumichi Hozumi (Mount Sinai Hospital ResearchInstitute). These 2H1 cells are conditional ras transformant thatexpress the H-ras oncogene in the presence of 50 μM ZnSO₄. All celllines were grown in Dulbecco's modified Eagle's medium (DMEM) containing10% fetal bovine serum (FBS).

[0026] The NIH-3T3 tet-myc cells were obtained from Dr. R. N. Johnston(University of Calgary) and were grown in DMEM containing 10%heat-inactivated FBS and antibiotics in the presence or absence of 2μg/ml tetracycline (Helbing, C. C. et al., Cancer Res. 57:1255-1258(1997)). In the presence of tetracycline, expression of the human c-mycgene is repressed. Removal of tetracycline results in the elevation ofexpression of c-myc by up to 100-fold in these cells, which also displaya transformed phenotype.

[0027] The PKR */* and PKR °/° mouse embryo fibroblasts (MEFs) wereobtained from Dr. B. R. G. Williams (the Cleveland Clinic Foundation)and were grown in α-MEM containing fetal bovine serum and antibiotics aspreviously described (Yang, Y. L. et al. EMBO J. 14:6095-6106 (1995);Der, S. D. et al., Proc. Natl. Acad. Sci. USA 94:3279-3283 (1997)).

[0028] The Dearing strain of reovirus serotype 3 used in these studieswas propagated in suspension cultures of L cells and purified accordingto Smith (Smith, R. E. et al., (1969) Virology, 39:791-800) with theexception that β-mercaptoethanol (β-ME) was omitted from the extractionbuffer. Reovirus labelled with [³⁵S]methionine was grown and purified asdescribed by McRae and Joklik (McRae, M. A. and Joklik, W. K., (1978)Virology, 89:578-593). The particle/PFU ratio for purified reovirus wastypically 100/1.

[0029] Immunofluorescent Analysis of Reovirus Infection

[0030] For the immunofluorescent studies the NIH-3T3, TNIH#S, H-ras,EJ-ras, 2H1 (+/−ZnSO₄), and THC-11 cells were grown on coverslips, andinfected with reovirus at a multiplicity of infection (MOI) of ˜10 PFUcell or mock-infected by application of the carrier agent(phosphate-buffered saline, PBS) to the cells in an identical fashion asthe administration of virus to the cells. At 48 hours postinfection,cells were fixed in an ethanol/acetic acid (20/1) mixture for 5 minutes,then rehydrated by sequential washes in 75%, 50% and 25% ethanol,followed by four washes with phosphate-buffered saline (PBS). The fixedand rehydrated cells were then exposed to the primary antibody (rabbitpolyclonal anti-reovirus type 3 serum diluted {fraction (1/100)} in PBS)[antiserum prepared by injection of rabbits with reovirus serotype 3 inFreund's complete adjuvant, and subsequent bleedings] for 2 hours atroom temperature. Following three washes with PBS, the cells wereexposed to the secondary antibody [goat anti-rabbit IgG (wholemolecule)-fluorescein isothiocyanate conjugate (FITC) [SigmaImmunoChemicals F-0382] diluted {fraction (1/100)} in PBS containing 10%goat serum and 0.005% Evan's Blue] for 1 hour at room temperature.Finally, the fixed and treated cells were washed three more times withPBS and then once with double-distilled water, dried and mounted onslides in 90% glycerol containing 0.1% phenylenediamine, and viewed witha Zeiss Axiophot microscope on which Carl Zeiss camera was mounted (themagnification for all pictures was 200×).

[0031] Detection of MAP Kinase (ERK) Activity

[0032] The PhosphoPlus p44/42 MAP kinase (Thr202/Tyr204) Antibody kit(New England Biolabs) was used for the detection of MAP kinase in celllysates according to the manufacturer's instructions. Briefly,subconfluent monolayer cultures were lysed with the recommendedSDS-containing sample buffer, and subjected to SDS-PAGE, followed byelectroblotting onto nitrocellulose paper. The membrane was then probedwith the primary antibody (anti-total MAPK or anti-phospho-MAPK),followed by the horseradish peroxidase (HRP)-conjugated secondaryantibody as described in the manufacturer's instruction manual.

[0033] Radiolabelling of Reovirus-Infected Cells and Preparation ofLysates

[0034] Confluent monolayers of NIH-3T3, TNIH#5, H-ras, EJ-ras, 2H1(+/−ZnSO₄), and THC-11 cells were infected with reovirus (MOI˜10PFU/cell). At 12 hours postinfection, the media was replaced withmethionine-free DMEM containing 10% dialyzed FBS and 0.1 mCi/ml[³⁵S]methionine. After further incubation for 36 hours at 37° C., thecells were washed in phosphate-buffered saline (PBS) and lysed in thesame buffer containing 11% Triton X-100, 0.5% sodium deoxycholate and 1mM EDTA. The nuclei were then removed by low speed centrifugation andthe supernatants were stored at −70° C. until use.

[0035] Preparation of Cytoplasmic Extracts for In Vitro Kinase Assays

[0036] Confluent monolayers of the various cell lines were grown on 96well cell culture plates. At the appropriate time postinfection themedia was aspirated off and the cells were lysed with a buffercontaining 20 mM HEPES [pH 7.4], 120 mM KCl, 5 mM MgCl₂, 1 mMdithiothreitol, 0.5% Nonidet P-40, 2 μg/ml leupeptin, and 50 μg/mlaprotinin. The nuclei were then removed by low-speed centrifugation andthe supernatants were stored at −70° C. until use.

[0037] Cytoplasmic extracts were normalized for protein concentrationsbefore use by the Bio-Rad protein microassay method. Each in vitrokinase reaction contained 20 μl of cell extract, 7.5 μl of reactionbuffer (20 mM HEPES [pH 7.4], 120 mM KCl, 5 mM MgCl₂, 1 mMdithiothreitol, and 10% glycerol) and 7.0 μl of ATP mixture (1.0μCi[γ-³²P]ATP in 7 μl of reaction buffer), and was incubated for 30minutes at 37° C. (Mundschau, L. J., and Faller, D. V., J. Biol. Chem.,267:23092-23098 (1992)). Immediately after incubation the labelledextracts were either boiled in Laemmli SDS-sample buffer or were eitherprecipitated with agarose-poly(I)poly(C) beads or immunoprecipitatedwith an anti-PKR antibody.

[0038] Agarose poly(I)poly(C) Precipitation

[0039] To each in vitro kinase reaction mixture, 30 μl of a 50% Agpoly(I)poly(C) Type 6 slurry (Pharmacia LKB Biotechnology) was added,and the mixture was incubated at 4° C. for 1 h. The Ag poly(I)poly(C)beads with the absorbed, labelled proteins were then washed four timeswith was buffer (20 mM HEPES [7.5 pH], 90 mM KCl, 0.1 mM EDTA, 2 mMdithiothreitol, 10% glycerol) at room temperature and mixed with2×Laemmli SDS sample buffer. The beads were then boiled for 5 min, andthe released proteins were analyzed by SDS-PAGE.

[0040] Polymerase Chain Reaction

[0041] Cells at various times postinfection were harvested andresuspended in ice cold TNE (10 mM Tris [pH 7.8], 150 mM NaCl, 1 mMEDTA) to which NP-40 was then added to a final concentration of 1%.After 5 minutes, the nuclei were pelleted and RNA was extracted from thesupernatant using the phenol:chloroform procedure. Equal amounts oftotal cellular RNA from each sample were then subjected to RT-PCR (Wong,H., et al, (1994) Anal. Biochem., 223:251-258) using randomhexanucleotide primers (Pharmacia) and RTase (GIBCO-BRL) according tothe manufacturers' protocol. The cDNA's from the RT-PCR step was thensubjected to selective amplification of reovirus s1 cDNA using theprimer 5′-AATTCGATTTAGGTGACACTATAGCTATTGGTCGGATG-3′ (SEQ ID NO:1) and5′-CCCTTTTGACAGTGATGCTCCGTTATCACTCG-3′ (SEQ ID NO:2) that amplify apredicted 116 bp fragment. These primer sequences were derived from theS1 sequence determined previously (Nagata, L., et al., (1984) NucleicAcids Res., 12:8699-8710). The GAPDH primers (Wong, H., et al., (1994)Anal. Biochem., 223:251-258), 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ (SEQ IDNO:3) and 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′ (SEQ ID NO:4) were used toamplify a predicted 306 bp GAPDH fragment which served as a PCR and gelloading control. Selective amplification of the s1 and GAPDH cDNA's wasperformed using Taq DNA polymerase (GIBCO-BRL) according to themanufacturers' protocol using a Perkin Elmer Gene Amp PCR system 9600.PCR was carried out for 28 cycles with each consisting of a denaturingstep for 30 seconds at 97° C., annealing step for 45 seconds at 55° C.,and polymerization step for 60 seconds at 72° C. PCR products wereanalyzed by electrophoresis through an ethidium bromide-impregnatedTAE-2% agarose gel and photographed under ultra-violet illumination withPolaroid 57 film.

[0042] Immunoprecipitation and SDS-PAGE Analysis

[0043] Immunoprecipitation of ³⁵S-labelled reovirus-infected celllysates with anti-reovirus serotype 3 serum was carried out aspreviously described (Lee, P. W. K. et al. (1981) Virology,108:134-146). Immunoprecipitation of ³²P-labelled cell lysates with ananti-PKR antibody (from Dr. Michael Mathews, Cold Spring Harbor) wassimilarly carried out. Immunoprecipitates were analyzed by discontinuousSDS-PAGE according to the protocol of Laemmli (Laernmli, U. K., (1970)Nature, 227:680-685).

Example 1 Activated Intermediates in the Ras Signalling Pathway AugmentReovirus Infection Efficiency

[0044] It was previously shown that 3T3 cells and their derivativeslacking epidermal growth factor receptors (EGFR) are poorly infectibleby reovirus, whereas the same cells transformed with either EGFR orv-erb B are highly susceptible as determined by cytopathic effects,viral protein synthesis, and virus output (Strong, J. E. et al., (1993)Virology, 197:405-411; Strong, J. E. and Lee, P. W. K, (1996) J. Virol.,70:612-616).

[0045] To determine whether downstream mediators of the EGFR signaltransduction pathway may be involved, a number of different NIH3T3-derived, transformed with constitutively activated oncogenesdownstream of the EGFR, were assayed for relative susceptibility toreovirus infection. Of particular interest were intermediates in the rassignalling pathway (reviewed by Barbacid, M., Annu. Rev. Biochem.,56:779-827 (1987); Cahill, M. A., et al., Curr. Biol., 6:16-19 (1996)).To investigate the Ras signalling pathway, NIH 3T3 parental cell linesand NIH 3T3 lines transfected with activated versions of Sos (Aronheirn,A., et al., (1994) Cell, 78:949-961) or ras (Mundschau, L. J. andFaller, D. V., (1992) J. Biol. Chem., 267:23092-23098) oncogenes wereexposed to reovirus, and their capacity to promote viral proteinsynthesis was compared.

[0046] Detection of viral proteins was initially carried out usingindirect immunofluorescent microscopy as described above. The resultsindicated that whereas the NIH 3T3 cells adopted a typically flattened,spread-out morphology with marked contact inhibition, the transformedcells all grew as spindle-shaped cells with much less contactinhibition. On comparing the uninfected parental cell lines with thevarious transformed cell lines, it was apparent that the morphology ofthe cells was quite distinct upon transformation. Upon challenge withreovirus, it became clear that parental NIH 3T3 line was poorlyinfectible (<5%), regardless of the source of the parental NIH 3T3 line.In contrast, the transfected cell lines each demonstrated relativelypronounced immunofluorescence by 48 hours postinfection (data notshown).

[0047] To demonstrate that viral protein synthesis was more efficient inthe Sos- or Ras-transformed cell lines, cells were continuously labeledwith [³⁵S]-methionine from 12 to 48 hr postinfection and the proteinswere analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), as described above.

[0048] The results showed clearly that the levels of viral proteinsynthesis were significantly higher in the Sos- or Ras-transformed cellsthan in parental NIH 3T3 cells. The identities of the viral bands wereconfirmed by immunoprecipitation of the labeled proteins with polyclonalanti-reovirus antibodies. Since the uninfected NIH 3T3 cells and theirtransformed counterparts displayed comparable levels of cellular proteinsynthesis and doubling times (data not shown), the observed differencein the level of viral protein synthesis could not be due to intrinsicdifferences in growth rates or translation efficiencies for these celllines.

[0049] The long-term fate of infected NIH-3T3 cells was followed bypassaging these cells for at least 4 weeks. They grew normally andappeared healthy, with no sign of lytic or persistent infection; novirus could be detected in the medium after this time (data not shown).

Example 2 Enhanced Infectibility Conferred by Activated Oncogenes is NotDue to Long-term Transformation or the Generalized Transformed State ofthe Cell

[0050] To determine whether the differences in susceptibility may be theresult of long-term effects of transformation, or the result of theactivated oncogene itself, a cell line expressing a zinc-induciblecellular Harvey-ras (c-H-ras) gene was tested for susceptibility toreovirus infectibility, as described above. These cells, called 2H 1,were derived from the C3H 10T1/2 cell line which is poorly infectible byreovirus (data not shown), and carry the c-H-ras gene under the controlof the mouse metallothionine-I promoter (Trimble, W. S. et al. (1986)Nature, 321:782-784).

[0051] Cells were either mock-treated or pretreated with 50 μM ZnSO₄ 18hours prior to infection or mock-infection (administration of carrieragent), followed by indirect immunofluorescent analysis of these cellsat 48 hours postinfection or mock-infection.

[0052] The results (not shown) demonstrated that uninduced cells werepoorly infectible (<5%) whereas those induced for only 18 hours weremuch more susceptible (>40%). Enhanced viral protein synthesis in theZn-induced 2H1 cells was further confirmed by metabolic labeling of thecells with [³⁵S]methionine followed by SDS-PAGE analysis ofvirus-specific proteins (not shown).

[0053] Based on these observations, the augmentation of reovirusinfection efficiency in the transformed cells is a direct result of theactivated oncogene product(s), and not due to other factors such asaneuploidy often associated with long-term transformation, or otheraccumulated mutations that may be acquired under a chronicallytransformed state (e.g., p53 or myc activation).

[0054] To show further that susceptibility to reovirus infection is nota result of transformation per se (i.e., a result of the transformedstate of the host cell), NIH-3T3 cells containing atetracycline-controlled human c-myc gene (tet-myc cells) were examined(Helbing, C. C. et al., Cancer Res. 57.1255-1258 (1997)). These cellsnormally are maintained in tetracycline (2 μg/ml) which represses theexpression of c-myc. Removal of tetracycline under normal growthconditions (10% fetal bovine serum) leads to accumulation of the c-Mycprotein and the cells display a transformed phenotype. We found thatthese cells were unable to support virus growth either in the presenceor in the absence of tetracycline (data not shown), suggesting thatsusceptibility to reovirus infection is not due to the generaltransformed state of the host cell, but rather requires specifictransformation by elements of the Ras signaling pathway.

[0055] A good indicator of activation of the Ras signaling pathway isthe activation of the MAP kinases ERK1 and ERK2 (for a review, seeRobinson, M. J. and Cobb, M. H., Curr. Opin. Cell. Biol 9:180-186(1997)). In this regard, it was found that, compared with untransformedcells, Ras-transformed cells have a significantly higher ERK1/2 activity(data not shown). Furthermore, an examination of a number of humancancer cell lines has revealed an escellent correlation between thelevel of ERK1/2 activity and susceptibility to reovirus infection (datanot shown), although ERK1/2 itself does not appear to play any role init. Mouse L cells and human HeLa cells, in which reovirus grows verywell, both manifest high ERK1/2 activity (data not shown).

Example 3 Viral Transcripts are Generated but Not Translated inReovirus-Resistant NIH 3T3 Cells

[0056] The step at which reovirus infection is blocked in nonsusceptibleNIH 3T3 cells was also identified. Because virus binding and virusinternalization for nonsusceptible cells were comparable to thoseobserved for susceptible cells (Strong, J. E. et al., (1993) Virology,197:405-411), the transcription of viral genes was investigated.

[0057] The relative amounts of reovirus S1 transcripts generated in NIH3T3 cells and the Ras-transformed cells during the first 12 hours ofinfection were compared after amplification of these transcripts bypolymerase chain reaction (PCR), as described above. The resultsdemonstrated that the rates of accumulation of S1 transcripts in the twocell lines were similar, at least up to 12 hours postinfection. Similardata were obtained when rates of accumulation of other reovirustranscripts were compared (data not shown). These results demonstratethat infection block in nonsusceptible cells is not at the level oftranscription of viral genes, but rather, at the level of translation Ofthe transcripts.

[0058] At later times, the level of viral transcripts present inuntransformed NIH-3T3 cells decreased significantly whereas transcriptsin transformed cells continued to accumulate (data not shown). Theinability of these transcripts to be translated in NIH-3T3 cellsprobably contributed to their degradation. As expected, the level ofviral transcripts in infected L cells was at least comparable with thatin infected Ras-transformed cells (data not shown).

Example 4 A 65 kDa Protein is Phosphorylated in Reovirus-treated NIH 3T3Cells but Not in Reovirus-Infected Transformed Cells

[0059] Because viral transcripts were generated, but not translated, inNIH 3T3 cells, it was investigated whether the double-stranded RNA(dsRNA)-activated kinase, PKR, is activated (phosphorylated) in thesecells (for example, by S1 mRNA transcripts which have been shown to bepotent activators of PKR ((Bischoff, J. R. and Samuel, C. E., (1989)Virology, 172:106-115), which in turn leads to inhibition of translationof viral genes. The corollary of such a scenario would be that in thecase of the transformed cells, this activation is prevented, allowingviral protein synthesis to ensue.

[0060] NIH 3T3 cells and v-erbB- or Ras-transformed cells (designatedTHC-11 and H-ras, respectively) were treated with reovirus (i.e.,infected) or mock-infected (as above), and at 48 hours post treatment,subjected to in vitro kinase reactions, followed by autoradiographicanalysis as described above.

[0061] The results clearly demonstrated that there was a distinctphosphoprotein migrating at approximately 65 kDa, the expected size ofPKR, only in the NIH 3T3 cells and only after exposure to reovirus. Thisprotein was not labeled in the lysates of either the uninfectedtransformed cell lines or the infected transformed cell lines. Instead,a protein migrating at approximately 100 kDa was found to be labeled inthe transformed cell lines after viral infection. This protein wasabsent in either the preinfection or the postinfection lysates of theNIH 3T3 cell line, and was not a reovirus protein because it did notreact with an anti-reovirus serum that precipitated all reovirusproteins (data not shown). A similar 100 kDa protein was also found tobe ³²P-labeled in in vitro kinase reactions of postinfection lysates ofthe Sos-transformed cell lines (data not shown).

[0062] That intermediates in the Ras signalling pathway were responsiblefor the lack of phosphorylation of the 65 kDa protein was furtherconfirmed by the use of the 2H1 cells which contain a Zn-inducible Rasoncogene. Uninduced 2H1 cells (relatively resistant to reovirusinfection, as shown above), were capable of producing the 65 kDaphosphoprotein only after exposure to virus. However, 2H1 cellssubjected to Zn-induction of the H-Ras oncogene showed significantimpairment of the production of this phosphoprotein. This impairmentcoincided with the enhancement of viral synthesis. These resultstherefore eliminated the possibility that the induction of the 65 kDaphosphoprotein was an NIH 3T3-specific event, and clearly establishedthe role of Ras in preventing (or reversing) induction of the productionof this phosphoprotein. The Zn-induced 2H1 cells did not produce the 100kDa phosphoprotein seen in the infected, chronically transformed H-Rascells.

Example 5 Induction of Phosphorylation of the 65 kDa Protein RequiresActive Viral Transcription

[0063] Since production of the 65 kDa phosphoprotein occurred only incells that were resistant to reovirus infection, and only after thecells were exposed to reovirus, it was investigated whether active viraltranscription was required for production of the 68 kDa phosphoprotein.Reovirus was UV-treated to inactivate its genome prior to administrationof the reovirus to NIH 3T3 cells. For UV-treatment, reovirus wassuspended in DMEM to a concentration of approximately 4×10⁸ PFU/mL andexposed to short-wave (254 nm) UV light for 20 minutes. UV-inactivatedvirus were non-infectious as determined by lack of cytopathic effects onmouse L-929 fibroblasts and lack of viral protein synthesis by methodsof [³⁵S]-methionine labelling as previously described. Such UV treatmentabolished viral gene transcription, as analyzed by PCR, and hence viralinfectivity (data not shown). The cells were then incubated for 48hours, and lysates were prepared and subjected to in vitro ³²P-labelingas before. The results showed that NIH 3T3 cells infected with untreatedreovirus produced a prominent 65 kDa ³²P-labelled band not found inuninfected cells. Cells exposed to UV-treated reovirus behaved similarlyto the uninfected control cells, manifesting little phosphorylation ofthe 65 kDa protein. Thus, induction of the phosphorylation of the 65 kDaphosphoprotein is not due to dsRNA present in the input reovirus;rather, it requires de novo transcription of the viral genes, consistentwith the identification of the 65 kDa phosphoprotein as PKR.

Example 6 Identification of the 65 kDa Phosphoprotein as PKR

[0064] To determine whether the 65 kDa phosphoprotein was PKR, a dsRNAbinding experiment was carried out in which poly(I)-poly(c) agarosebeads were added to ³²P labeled lysates, as described above. Afterincubation for 30 minutes at 4° C., the beads were washed, and boundproteins were released and analyzed by SDS-PAGE. The results showed thatthe 65 kDa phosphoprotein produced in the postinfection NIH 3T3 celllysates was capable of binding to dsRNA; such binding is awell-recognized characteristic of PKR. In contrast, the 100 kDaphosphoprotein detected in the infected H-ras-transformed cell line didnot bind to the Poly(I)-poly(c) agarose. The 65 kDa phosphoprotein wasalso immunoprecipitable with a PKR-specific antibody (provided by Dr.Mike Mathews, Cold Spring Harbor Laboratory), confirming that it wasindeed PKR.

Example 7 PKR Inactivation or Deletion Results in Enhanced Infectibilityof Untransformed Cells

[0065] If PKR phosphorylation is responsible for the shut-off of viralgene translation in NIH-3T3 cells, and one of the functions of theactivated oncogene product(s) in the transformed cells is the preventionof this phosphorylation event, then inhibition of PKR phosphorylation inNIH-3T3 cells by other means (e.g. drugs) should result in theenhancement of viral protein synthesis, and hence infection, in thesecells. To test this idea, 2-aminopurine was used. This drug has beenshown to possess relatively specific inhibitory activity towards PKRautophosphorylation (Samuel, C. E. and Brody, M., (1990) Virology,176:106-113; Hu, Y. and Conway, T. W. (1993), J Interferon Res.,13:323-328). Accordingly, NIH 3T3 cells were exposed to 5 mM2-aminopurine concurrently with exposure to reovirus. The cells werelabeled with [³⁵]methionine from 12 to 48 h postinfection, and lysateswere harvested and analyzed by SDS-PAGE.

[0066] The results demonstrated that exposure to 2-aminopurine resultedin a significantly higher level of viral protein synthesis in NIH 3T3cells (not shown). The enhancement was particularly pronounced afterimmunoprecipitating the lysates with an anti-reovirus serum. Theseresults demonstrate that PKR phosphorylation leads to inhibition ofviral gene translation, and that inhibition of this phosphorylationevent releases the translation block. Therefore, intermediates in theRas signalling pathway negatively regulate PKR, leading to enhancedinfectibility of Ras-transformed cells.

[0067] Interferon β, known to induce PKR expression, was found tosignificantly reduce reovirus replication in Ras-transformed cells (datanot shown).

[0068] A more direct approach to defining the role of PKR in reovirusinfection is through the use of cells that are devoid of PKR.Accordingly, primary embryo fibroblasts from wild-type PKR */* and PKR°/° mice (Yang, Y. L. et al. EMBO J. 14:6095-6106 (1995)) were comparedin terms of susceptibility to reovirus infection. 110 The resultsclearly showed that reovirus proteins were synthesized at asignificantly higher level in the PKR °/° cells than in the PKR+/cells.These experiments demonstrated that PKR inactivation or deletionenhanced host cell susceptibility to reovirus infection in the same wayas does transformation by Ras or elements of the Ras signaling pathway,thereby providing strong support of the role of elements of the Rassignaling pathway in negatively regulating PKR.

Example 8 Inactivation of PKR in Transformed Cells Does Not Involve MEK

[0069] Receptor tyrosine kinases such as EGFRs are known to stimulatethe mitogen-activated or extracellular signal-regulated kinases (ERK1/2)via Ras (see Robinson, M. J. and Cobb, M. H., Curr. Opin. Cell. Biol.9:180-186 (1997)). This stimulation requires the phosphorylation ofERK1/2 by the mitogen-activated extracellular signal-regulated kinase,ldnase MEK, which itself is activated (phosphorylated) by Raf, aserine-threonine kinase downstream of Ras. To determine if MEK activitywas required for PKR inactivation in transformed cells, the effect ofthe recently identified EK inhibitor PD98059 (Dudley, D. T. et al.,Proc. Natl. Acad. Sci. USA 92:7686-7689 (1995); Waters, S. D. et al., J.Biol. Chem. 270:20883-20886 (1995)) on infected Ras-transformed cellswas studied.

[0070] H-Ras-transformed cells were grown to 80% confluency and infectedwith reovirus at an m.o.i. of approximately 10 p.f.u./cell. PD98059(Calbiochem), dissolved in dimethylsulfoxide (DMSO), was applied to thecells at the same time as the virus (final concentration of PD98059 was50 μM). The control cells received an equivalent volume of DMSO. Thecells were labeled with ³⁵S-methionine from 12 to 48 hourspost-infection. Lysates were then prepared, immunoprecipitated with thepolyclonal anti-reovirus serotype 3 serum and analyzed by SDS-PAGE.

[0071] The results (data not shown) showed that PD98059, at aconcentration that effectively inhibited ERK1/2 phosphorylation, did notinhibit reovirus protein synthesis in the transformed cells. On thecontrary, PD98059 treatment consistently caused a slight enhancement ofviral protein synthesis in these cells; the reason for this is underinvestigation. Consistent with the lack of inhibition of viral proteinsynthesis in the presence of PD98059, the PKR in these cells remainedunphosphorylated (data not shown). As expected, PD98059 had no effect onreovirus-induced PKR phosphorylation in untransformed NIH-3T3 cells(data not shown). These results indicated that MEK and ERK1/2 are notinvolved in PKR activation.

Example 9 In Vivo Oncolytic Capability of Reovirus

[0072] A severe combined immunodeficiency (SCID) host tumor model wasused to assess the efficacy of utilizing reovirus for tumorreductionMale and female SCID mice (Charles River, Canada) were injected withv-erbB-transformed NIH 3T3 mouse fibroblasts (designated THC-11 cells)in two subcutaneous sites overlying the hind flanks. In a first trial,an injection bolus of 2.3×10⁵ cells in 100 μl of sterile PBS was used.In a second trial, an injection bolus of 4.8×10⁶ cells in 100 μl PBS wasused. Palpable tumors were evident approximately two to three weeks postinjection.

[0073] Reovirus serotype three (strain Dearing) was injected into theright-side tumor mass (the “treated tumor mass”) in a volume of 20 μl ata concentration of 1.0×10⁹ plaque forming units (PFU)/ml. The left-sidetumor mass (the “untreated tumor mass”) was left untreated. The micewere observed for a period of seven days following injection withreovirus, measurements of tumor size were taken every two days usingcalipers, and weight of tumors was measured after sacrifice of theanimals. All mice were sacrificed on the seventh day. Results are shownin Table 1. TABLE 1 Tumor Mass after Treatment with Reovirus Trial 1 (n= 8) mean untreated tumor mass   602 mg mean treated tumor mass   284 mgTrial 2 (n = 12) mean control tumor mass 1523.5 mg mean untreated tumormass  720.9 mg mean treated tumor mass  228.0 mg

[0074] The treated tumor mass was 47% of that of the untreated tumormass in trial 1, and 31.6% of the untreated tumor mass in trial 2. Theseresults indicated that the virus-treated tumors were substantiallysmaller than the untreated tumors, and that there may be an additionalsystemic effect of the virus on the untreated tumor mass.

[0075] Similar experiments were also conducted using unilateralintroduction of tumor cells. SCID mice were injected subcutaneously andunilaterally in the hind flank with v-erbB-transformed NIH 3T3 mousefibroblasts (THC-11 cells). Palpable tumors (mean area 0.31 cm²) wereestablished after two weeks. Eight animals were then given a singleintratumoral injection of 1.0×10⁷ PFUs of reovirus serotype 3 (strainDearing) in phosphate-buffered saline (PBD). Control tumors (n=10) wereinjected with equivalent amounts of UV-inactivated virus. Tumor growthwas followed for 12 days, during which time no additional reovirustreatment was administered.

[0076] Results, shown in FIG. 2, demonstrated that treatment of thesetumors with a single dose of active reovirus (open circles) resulted indramatic repression of tumor growth by the thirteenth day (endpoint),when tumors in the control animals injected with a single dose ofinactivated reovirus (closed circles) exceeded the acceptable tumorburden. This experiment was repeated several times and found to behighly reproducible, thus further demonstrating the efficacy of reovirusin repressing tumor growth.

Example 10 In Vivo Oncolytic Capability of Reovirus Against Human BreastCancer-Derived Cell Lines

[0077] In vivo studies were also carried out using human breastcarcinoma cells in a SCID mouse model. Female SCID mice were injectedwith 1×10⁹ MDAMB468 cells in two subcutaneous sites, overlying both hindflanks. Palpable tumors were evident approximately two to four weekspost injection. Undiluted reovirus serotype three (strain Dearing) wasinjected into the right side tumor mass in a volume of 20 μl at aconcentration of 1.0×10⁹ PFU/ml. The results are shown in Table 2. TABLE2 Tumor Mass After Treatment with Reovirus mean untreated tumor meantreated tumor TREATMENT mass (left side) mass (right side) Reovirus (N =8) 29.02 g  38.33 g Control (N = 8) 171.8 g 128.54 g

[0078] Although these studies were preliminary, it was clear that thesize of the tumors in the reovirus-treated animals was substantiallylower than that in the untreated animals. However, the size of thetumors on the right (treated) side of the reovirus-treated animals wasslightly larger on average than the left (untreated) side. This wasunexpected but may be explained by the composition of the mass beingtaken up by inflammatory cells with subsequent fibrosis, as well as bythe fact that these tumors were originally larger on the right side onaverage than the left. The histologic composition of the tumor masses isbeing investigated. These results also support the systemic effect thereovirus has on the size of the untreated tumor on the contralateralslide of reovirus injection.

Example 11 Susceptibility of Additional Human Tumors to ReovirusOncolysis

[0079] In view of the in vivo results presented above, the oncolyticcapability observed in murine cells was investigated in cell linesderived from additional human tumors.

[0080] A. Materials and Methods

[0081] Cells and Virus

[0082] All cell lines were grown in Dulbecco's modified Eagle's medium(DMEM) containing 10% fetal bovine serum (FBS).

[0083] The Dearing strain of reovirus serotype 3 used in these studieswas propagated in suspension cultures of L cells and purified accordingto Smith (Smith, R. E. et al., (1969) Virology, 39:791-800) with theexception that P-mercaptoethanol (p-ME) was omitted from the extractionbuffer. Reovirus labelled with [³⁵S]methionine was grown and purified asdescribed by McRae and Joklik (McRae, M. A. and Joklik, W. K., (1978)Virology, 89:578-593). The particle/PFU ration for purified reovirus wastypically 100/1.

[0084] Cytopathic Effects of Reovirus on Cells

[0085] Confluent monolayers of cells were infected with reovirusserotype 3 (strain Dearing) at a multiplicity of infection (MOI) ofapproximately 40 plaque forming units (PFU) per cell. Pictures weretaken at 36 hour postinfection for both reovirus-infected andmock-infected cells.

[0086] Immunofluorescent Analysis of Reovirus Infection

[0087] For the immunofluorescent studies the cells were grown oncoverslips, and infected with reovirus at a multiplicity of infection(MOI) of10 PFU/cell or mock-infected as described above. At varioustimes postinfection, cells were fixed in an ethanol/acetic acid (20/1)mixture for 5 minutes, then rehydrated by subsequential washes in 75%,50% and 25% ethanol, followed by 4 washes with phosphate-buffered saline(PBS). The fixed and rehydrated cells were then exposed to the primaryantibody (rabbit polyclonal anti-reovirus type 3 serum diluted {fraction(1/100)} in PBS) for 2 hr at room temperature. Following 3 washes withPBS, the cells were exposed to the secondary antibody [goat anti-rabbitIgG (whole molecule) fluorescein isothiocyanate (FITC) conjugate diluted{fraction (1/100)} in PBS containing 10% goat serum and 0.005% Evan'sBlue counterstain] for 1 hour at room temperature. Finally, the fixedand treated cells were washed 3 more times with PBS, followed by 1 washwith double-distilled water, dried and mounted on slides in 90% glycerolcontaining 0.1% phenylenediamine, and viewed with a Zeiss Axiophotmicroscope mounted with a Carl Zeiss camera (magnification for allpictures was 200×).

[0088] Infection of cells and quantitation of virus

[0089] Confluent monolayers of cells grown in 24-well plates wereinfected with reovirus at an estimated multiplicity of 10 PFU/cell.After 1 hour incubation at 37° C., the monolayers were washed with warmDMEM-10% FBS, and then incubated in the same medium. At various timespostinfection, a mixture of NP-40 and sodium deoxycholate was addeddirectly to the medium on the infected monolayers to finalconcentrations of 1% and 0.5%, respectively. The lysates were thenharvested and virus yields were determined by plaque titration on L-929cells.

[0090] Radiolabelling of Reovirus-Infected Cells and Preparation ofLysates

[0091] Confluent monolayers of cells were infected with reovirus (MOI˜10PFU/cell). At various times postinfection, the media was replaced withmethionine-free DMEM containing 10% dialyzed PBS and 0.1 mCi/ml[³⁵S]methionine. After further incubation for 1 hour at 37° C., thecells were washed in phosphate-buffered saline (PBS) and lysed in thesame buffer containing 1% Triton X-100, 0.5% sodium deoxycholate and 1mM EDTA. The nuclei were then removed by low speed centrifugation andthe supernatants was stored at 70° C. until use.

[0092] Immunoprecipitation and SDS-PAGE Analysis

[0093] Immunoprecipitation of [⁵S]-labelled reovirus-infected celllysates with anti-reovirus serotype 3 serum was carried out aspreviously described (Lee, P. W. K. et al. (1981) Virology,108:134-146). Immunoprecipitates were analyzed by discontinuous SDS-PAGEaccording to the protocol of Laemrnli (Laemmli, U. K, (1970) Nature,227:680-685).

[0094] B. Breast Cancer

[0095] The c-erbB-2/neu gene encodes a transmembrane protein withextensive homology to the EGFR that is overexpressed in 20-30% ofpatients with breast cancer (Yu, D. et al. (1996) Oncogene 13:1359).Since it has been established herein that Ras activation, either throughpoint mutations or through augmented signaling cascade elements upstreamof Ras (including the c-erbB-2/neu homologue EGFR) ultimately creates ahospitable environment for reovirus replication, an array of cell linesderived from human breast cancers were assayed for reovirussusceptibility. The cell lines included MDA-MD-435SD (ATCC depositHTB-129), MCF-7 (ATCC deposit HTB-22), T-27-D (ATCC deposit HTB-133),BT-20 (ATCC deposit HTB-19), HBL-100 (ATCC deposit HTB-124), MDA-MB468(ATCC deposit HTB-132), and SKBR-3 (ATCC deposit HTB-30).

[0096] Based upon induction of cytopathic effects, and viral proteinsynthesis as measured by radioactive metabolic labeling andimmunofluorescence as described above, it was found that five out ofseven of the tested breast cancers were susceptible to reovirusinfection: MDA-MB435S, MCF-7, T-27-D, MDA MB468, and SKBR-3 wereexquisitely sensitive to infection, while BT-20 and HBL-100 demonstratedno infectibility.

[0097] C. Brain Glioblastoma

[0098] Next a variety of cell lines derived from human brainglioblastomas was investigated. The cell lines included A-172, U-118,U-178, U-563, U-251, U-87 and U-373 (cells were a generous gift from Dr.Wee Yong, University of Calgary).

[0099] Six out of seven glioblastoma cell lines demonstratedsusceptibility to reovirus infection, including U-118, U-178, U-563,U-251, U-87 and U-373, while A-172 did not demonstrate anyinfectibility, as measured by cytopathic effects, immunofluorescence and[³⁵S]-methionine labeling of reovirus proteins.

[0100] The U-87 glioblastoma cell line was investigated further. Toassess the sensitivity of U-87 cells to reovirus, U-87 cells (obtainedfrom Dr. Wee Yong, University of Calgary) were grown to 80% confluencyand were then challenged with reovirus at a multiplicity of infection(MOI) of 10. Within a period of 48 hours there was a dramatic,widespread cytopathic effect (data not shown). To demonstrate furtherthat the lysis of these cells was due to replication of reovirus, thecells were then pulse-labeled with [³⁵S]methionine for three hourperiods at various times postinfection and proteins were analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) asdescribed above. The results (not shown) clearly demonstrated effectivereovirus replication within these cells with resultant shutoff of hostprotein synthesis by 24 hours postinfection.

[0101] The U-87 cells were also introduced as human tumor xenograftsinto the hind flank of 10 SCID mice. U-87 cells were grown in Dulbecco'smodified Eagle's medium containing 10% fetal bovine serum, as describedabove. Cells were harvested, washed, and resuspended in sterile PBS;2.0×10⁶ cells in 100 μl were injected subcutaneously at a site overlyingthe hind flank in five- to eight-week old male SCID ice (Charles River,Canada). Tumor growth was measured twice weekly for a period of fourweeks. Results, shown in FIG. 3, demonstrated that treatment of U-87tumors with a single intratumoral injection of 1.0×10⁷ PFUs of livereovirus (open circles, n=5) resulted in drastic repression of tumorgrowth, including tumor regression by the fourth week post-treatment(P=0.008), in comparison with treatment of tumors with a singleintraturnoral injection of the same amount of UV-inactivated reovirus(closed circles, n=5).

[0102] Hematoxylin/eosin (HE)-staining of the remaining microfoci of thetumors treated with active virus, performed as described (H. Lyon, CellBiology, A Laboratory Handbook, J. E. Celis, ed., Academic Press, 1994,p. 232) revealed that the remaining tumor mass consisted largely ofnormal stroma without appreciable numbers of viable tumor cells, nor wasthere any eevidence of infiltration of tumor cells into the underlyingsceletal muscle (data not shown). Necrosis of tumor cells was due todirect lysis by the virus, the same mechanism of cell killing as byreovirus in vitro.

[0103] To determine if there was viral spread beyond the tumor mass,immunofluorescent microscopy using antibodies directed against totalreovirus proteins was conducted as described above, on sections of thetumor and adjoining tissue. It was found that reovirus-specific proteinswere confined to the tumor mass; no viral staining was detected in theunderlying skeletal muscle (data not shown). As expected, viral proteinswere not present in tumors injected with the UV-inactivated virus (datanot shown). These results demonstrated that reovirus replication inthese animals was highly tumor specific with viral amplification only inthe target U-87 cells.

[0104] Since most tumors are highly vascularized, it was likely thatsome virus could enter the blood stream following the lysis of theinfected tumor cells. To determine if there was systemic spread of thevirus, blood was harvested from the treated and control animals, andserially diluted for subsequent plaque titration. Infectious virus wasfound to be present in the blood at a concentration of 1×10⁵ PFUs/ml(data not shown).

[0105] The high degree of tumor specificity of the virus, combined withsystemic spread, suggested that reovirus could be able to replicate inglioblastoma tumors remote from the initially infected tumor, asdemonstrated above with regard to breast cancer cells. To verify thishypothesis, SCID mice were implanted bilaterally with U-87 human tumorxenografts on sites overlying each hind flank of the animals. Thesetumors were allowed to grow until they measured 0.5×0.5 cm. Theleft-side tumors were then administered a single infection of reovirusin treated animals (n=5); control animals (n=7) were mock-treated withUV-inactivated virus. Tumors were again measured twice weekly for aperiod of four weeks.

[0106] Results, shown in FIG. 4, demonstrated that inhibition andeventual regression of both the treated (circles) and untreated tumormasses (squares) occurred only in the live reovirus-treated animals(open circles and squares), in contrast with the inactivatedreovirus-treated animals (closed circles and squares). Subsequentimmunofluorescent analysis revealed that reovirus proteins were presentin both the ipsilateral (treated) as well as the contralateral(untreated) tumor, indicating that regression on the untreated side wasa result of reovirus oncolysis (data not shown).

[0107] D. Pancreatic Carcinoma

[0108] Cell lines derived from pancreatic cancer were investigated fortheir susceptibility to reovirus infection. The cell lines includedCapan-1 (ATCC deposit HTB-79), BxPC3 (ATCC deposit CRL-1687), MIAPACA-2(ATCC deposit CRL-1420), PANC-1 (ATCC deposit CRL-1469), AsPC-1 (ATCCdeposit CRL-1682) and Hs766T (ATCC deposit HTB-134).

[0109] Five of these six cell lines demonstrated susceptibility toreovirus infection including Capan-1, MIAPACA-2, PANC-1, AsPC-1 andHs766T, whereas BxPC3 demonstrated little infectability as assayed byvirus-induced cytopathological effects, immunofluorescence and[³⁵S]-labelling. Interestingly, four of the five cell linesdemonstrating susceptibility to reovims oncolysis have been shown topossess transforming mutations in codon 12 of the K-ras gene (Capan-1,MLAPACA-2, PANC-1 and AsPC-1) whereas the one lacking suchsusceptibility (BxPC3) has been shown to lack such a mutation (Berrozpe,G., et al. (1994), Int. J. Cancer, 58:185-191). The status of the otherK-ras codons is currently unknown for the Hs766T cell line.

Example 12 Use of Reovirus as an Oncolytic Agent in Immune-CompetentAnimals

[0110] A syngeneic mouse model was developed to investigate use ofreovirus in immune-competent animals rather than in SCID mice asdescribed above. C3H mice (Charles River) were implanted subcutaneouslywith 1.0×10⁷ PFUs ras-transformed C3H cells (a gift of D. Edwards,University of Calgary). Following tumor establishment, mice were treatedwith a series of injections of either live reovirus (1.0×10⁸ PFUs) orUV-inactivated reovirus. Following an initial series (six injectionsover a nine-day course), test animals received a treatment of dilutereovirus (1.0×10⁷ PFUs) every second day. Mock-treated animals receivedan equivalent amount of V-inactivated virus.

[0111] Results demonstrated that reovirus was an effective oncolyticagent in these immune competent animals. All of the test animals showedregression of tumors; 5 of the 9 test animals exhibited complete tumorregression after 22 days, a point at which the control animals exceededacceptable tumor burden. Furthermore, there were no identifiable sideeffects in the animals treated with reovirus.

[0112] To assess the effects of previous reovirus exposure on tumorrepression and regression, one-half of a test group was challenged withreovirus (intramuscular injection of 1.0×10 ⁸ PFUs, type 3 Dearing)prior to tumor establishment. Two weeks after challenge, neutralizingantibodies could be detected in all exposed animals. Following tumorestablishment, animals were treated with a series of either live orUV-inactivated reovirus, as described above.

[0113] Results (data not shown) demonstrated that animals withcirculating neutralizing antibodies to reovirus (i.e., those challengedwith reovirus prior to tumor establishment) exhibited tumor repressionand regression similar to those animals in which there was no priorexposure to reovirus. Thus, reovirus can serve as an effective oncolyticagent even in immune-competent animals with previous exposure toreovirus.

[0114] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims.

What is claimed is:
 1. Use of a reovirus for the manufacture of a medicament for treating a Ras-mediated neoplasm in a mammal.
 2. The use of claim 1, wherein the reovirus is a human reovirus.
 3. The use of claim 2, wherein the human reovirus is selected from the group consisting of: type 1 reovirus, type 2 reovirus, and type 3 reovirus.
 4. The use of claim 1, wherein the reovirus is a nonhuman reovirus.
 5. The use of claim 4, wherein the nonhuman reovirus is selected from the group consisting of: mammalian reovirus and avian reovirus.
 6. The use of claim 1, wherein more than one type of reovirus is administered.
 7. The use of claim 1, wherein more than one strain of reovirus is administered.
 8. The use of claim 1, wherein the reovirus is a field isolate.
 9. The use of claim 1, wherein the reovirus is treated with a protease prior to administration.
 10. The use of claim 1, wherein the neoplasm is a solid neoplasm.
 11. The use of claim 1, wherein the neoplasm is a hematopoietic neoplasm.
 12. The use of claim 1, wherein the mammal is selected from the group consisting of: mice, dogs, cats, sheep, goats, cows, horses, pigs, and non-human primates.
 13. The use of claim 1, wherein the mammal is a human.
 14. The use of claim 1, wherein the neoplasm is selected from the group consisting of: pancreatic cancer, breast cancer and brain cancer.
 15. The use of claim 1, wherein the neoplasm is selected from the group consisting of: lung cancer, prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, and leukemia.
 16. The use of claim 1, wherein the ras-mediated neoplasm is metastatic.
 17. A method of treating a Ras-mediated neoplasm in a mammal, comprising administering to the neoplasm a reovirus in an amount sufficient to result in reovirus-mediated oncolysis of cells of the neoplasm.
 18. The method of claim 17, wherein the reovirus is a human reovirus.
 19. The method of claim 18, wherein the human reovirus is selected from the group consisting of: type 1 reovirus, type 2 reovirus, and type 3 reovirus.
 20. The method of claim 16, wherein the reovirus is a non-human reovirus.
 21. The method of claim 20, wherein the reovirus is selected from the group consisting of: mammalian reovirus and avian reovirus.
 22. The method of claim 17, wherein more than one type of reovirus is administered.
 23. The method of claim 17, wherein more than one strain of reovirus is administered.
 24. The method of claim 17, wherein the reovirus is a field isolate.
 25. The method of claim 17, wherein the reovirus is treated with a protease prior to administration.
 26. The method of claim 17, wherein the neoplasm is a solid neoplasm.
 27. The method of claim 26, wherein the reovirus is administered by injection into the solid neoplasm.
 28. The method of claim 26, wherein the reovirus is administered intravenously into the mammal.
 29. The method of claim 17, wherein the neoplasm is a hematopoietic neoplasm.
 30. The method of claim 29, wherein the reovirus is administered intravenously into the mammal.
 31. The method of claim 29, wherein the reovirus is administered intraperitoneally into the mammal.
 32. The method of claim 17, wherein the mammal is selected from the group consisting of: mice, dogs, cats, sheep, goats, cows, horses, pigs, and non-human primates.
 33. The method of claim 17, wherein the mammal is a human.
 34. The method of claim 17, wherein the neoplasm is selected from the group consisting of: pancreatic cancer, breast cancer and brain cancer.
 35. The method of claim 17, wherein the neoplasm is selected from the group consisting of: lung cancer, prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, and leukemia.
 36. The method of claim 17, wherein approximately 103 to 1012 plaque forming units of reovirus are administered.
 37. The method of claim 17, wherein the reovirus is administered in a single dose.
 38. The method of claim 17, wherein the reovirus is administered in more than one dose.
 39. The method of claim 17, wherein the reovirus is administered to more than one neoplasm in the mammal.
 40. The method of claim 17, wherein the ras-mediated neoplasm is metastatic.
 41. The method of claim 40, wherein the reovirus is administered to a single solid neoplasm.
 42. The method of claim 40, wherein the reovirus is administered intravenously.
 43. A method of treating a Ras-mediated neoplasm in a human, comprising administering to the neoplasm a reovirus in an amount sufficient to result in reovirus-mediated oncolysis of cells of the neoplasm. 